Mucopolysaccharide composition having a regulatory action on coagulation, medicament process for preparation and method of use

ABSTRACT

The invention pertains to a mucopolysaccharide fraction obtainable from heparin or from fractions including heparinic constituents of molecular weights from 2,000 to 50,000, which has a Yin-Wessler titer which is high relative to the USP titer. It contains components whose molecular weights are less than 10,000, particularly oligosaccharides in the area of 2,000-3,000, comprising from 8 to 12, notably 10 monosaccharide units, among which glucosamine units whose primary positions are sulphated. The last mentioned oligosaccharides include one N-acetyl-glucosamine unit per two units of 2-0-sulphate iduronic acid and per two N-sulphate-glucosamine units, the other saccharide units being of a different nature and including distinct substituents.

This application is a continuation-in-part application of our co-pendingapplication Ser. No. 204,505 pending filed Nov. 6, 1980, which was acontinuation of the co-pending application Ser. No. 091,164 nowabandoned filed Nov. 5, 1979.

The invention relates to a mucopolysaccharide fraction endowed withbiological properties, enabling it notably to play a regulator role withrespect to blood coagulation. Such a fraction can notably be obtainedfrom heparin preparations, such as mammalian tissue extracts.

Certainly heparin is doubtless until now one of the most importantanticoagulant medicaments, if not the most important, available to theclinician. It is in fact capable of taking part at several levels incascades of successive enzymatic reactions, which are normally engagedin the course of physiological hemostasis, in any situation capable ofresulting in hypercoagulability of the blood. It is more particularlycapable of simultaneously depressing a large number of the coagulationfactors participating to the creation and the up keeping of differentforms of hypercoagulability.

There will be recalled below, within the limits necessary for clarity ofthe description, some of the basic notions, purposely expresslysimplified, relating to coagulation. The coagulation process comprisesin fact three phases generally described as successive, even if they areintrically interrelated:

thromboplastin formation, the phase of prothrombinase (or activethromboplastin) formation,

thrombin formation, which phase can be summarized as the conversion ofthe prothrombin into thrombin under the influence of prothrombinase inthe presence of ionized calcium and finally,

fibrin formation, the phase in the course of which the blood fibrogenis, under the effect of the thrombin, converted into fibrin, whichprotein tends to become insoluble.

The formation of prothrombinase occurs, in the course of thethromboplastin formation step essentially according to two differentroutes: the intrinsic or endogenic route, and the extrinsic or exogenicroute, which end in the formation of prothrombinases of respectivelyplasmatic and tissular origins, both capable of activating prothrombininto active thrombin.

The intrinsic or endogenic route (or system) involves a large number offactors or plasmatic proenzymes capable of being successively activated(factors XII, XI, IX, VIII and X), where each activated product (factorsXIIa, XIa, IXa, VIIIa and Xa) acts like an enzyme capable of activatingthe following proenzyme, the activated X factor (Xa) then taking part,notably by reaction with the V factor and a phospholipid of plateletorigin, in the production of active endogenic plasmatic prothrombinase.The extrinsic or exogenic system, which can notably be directlydependent of a tissular lesion, calls upon a more limited number offactors and includes notably the production of tissular thromboplastinwhich, in combination with the VII factor, can, just as the factorVIIIa, convert the inactive X factor into the Xa factor. The activationsequence of the prothrombin into thrombin is then substantially the sameas for the intrinsic system, but the phospholipid is here of tissularand not of plasmatic origin.

It is hence possible, to some limited extent to express the idea thatthe two intrinsic and extrinsic routes join each other at the level ofthe activation of the X factor (also called Stuart factor), the twofollowing phases of coagulation--thrombin formation and fibrinformation, no longer then giving rise to a distinction between theintrinsic and extrinsic routes.

The outcome of the coagulation process consists in the formation of aninsoluble fibrin clot, intended notably to fill in the lesion at theorigin of the triggering of this process, for example at the level of ablood vessel.

These coagulation processes normally give rise then to a process, calledfibrinolysis, intended to produce lysis of the clot, notably under theeffect of plasmin, which enzyme only exists normally in the circulatingblood in the form of an inactive precursor, plasminogen, the fibrinitself constituting nonetheless one of the factors capable of initiatingthe conversion of the inactive plasminogen into fibrinolytically activeplasmin.

In fact, although there has, in the foregoing, been presented systems ofcoagulation and of fibrinolysis as two processes occurring successivelyin time, it is still not normally so in reality. In fact, there areinvolved balanced mechanisms, according to extremely complex processes,under the dependence of harmoniously opposed activator and inhibitorfactors. The unbalance of these mechanisms, in the sense ofhypercoagulability, is then capable of resulting in thromboses. On theother hand, a disequilibrium in the sense of hypocoagulability, exposesthe host to hemorragic risks.

It is obviously to palliate the effects of hypercoagulability thatrecourse is currently had to the powerful anticoagulant properties ofheparin, in order to bring back the coagulation-fibrinolysis mechanismto equilibrium, each time that the latter is subjected to a considerabledisturbance, for example on the occasion of a surgical operation on thehost. It is however well known that these attempts at re-equilibrationare extremely delicate and that, consequently, the administration of toohigh a dose of anticoagulant medicament--or the insufficient selectivityof the latter--for the purpose of preventing the risks ofhypercoagulation, for example the appearance of post-operativethromboses, may finally be at the origin of serious hemorragies: whencethe necessity of constant watch of the treated patients and of thenecessary adjustments of the doses administered--continuously ordiscontinuously--according to the results of tests, notably of overallcoagulability, like the Howell time, which must be practiced at regularintervals.

It is hence an object of the invention to provide active principles ofmedicaments (and the medicaments themselves) capable of overcoming thesedifficulties at least in part, notably which are capable of permitting apossible re-equilibration and/or easier control, at the cost of a lesserclinical watch of the coagulation-fibrinolysis system in patientsafflicted with a pathology of the coagulation which have undergone atreatment, such as a surgical operation, which expose them to risks ofhypercoagulability.

The invention relates more particularly to a mucopolysaccharide fractionexerting a regulator effect with regard to coagulation, notably incausing it to be delayed, yet upon bringing into play inhibitoryactionswhich are more selective than those of heparin, with respect to asmaller number of coagulation factors, more particularly with respect tothe activated X factor.

The invention hence relates to a mucopolysaccharide fraction obtainablefrom heparin or from fractions including heparinic constituents ofmolecular weights extending notably from about 2,000 to 50,000, such asobtained by extraction from mammalian tissues, this fraction beingcharacterized in that it is soluble in an aqueous-alcoholic medium(water-ethanol) having a titer of 55°-61° GL, in that it tends toinsolubility in a water-ethanol medium having a higher alcohol content,in that it is insoluble in pure alcohol, and in that it has aYin-Wessler titer and a USP titer which are respectively in a ratio atleast equal to 2, notably at least 3, preferably higher than 6.

These mucopolysaccharide fractions give rise to supplementaryfractionations, enabling the preparation of mucopolysaccharide fractionsof high specific activity, at the level of the Yin-Wessler titer andhaving ratios of the Yin-Wessler titer to the USP titer exceeding 10,even 16.

The Yin-Wessler titer is measured by the technique of these authorswhich is described in "J. Lab. Clin. Med.", 1976, 81, 298-300.

The USP titer, which measures, in manner known in itself, an overallcoagulation intensity under well determined conditions, is well known.It is recalled that it is determined in the manner described in thePharmacopea of the United States XIX, pp. 229-230, (see also the SecondSupplement USP-NF, p.62, and the Fourth Supplement USP-NF, p. 90,respectively entitled "Drug Substances and Dosage Forms".

The invention provides a particularly interesting active principle owingto the capacity that it has of inhibiting the Xa factor in a mannerwhich may be very selective, which capacity contrasts with its activityon the overall coagulation, which may be maintained at a very low level.

This mucopolysaccharide fraction hence constitutes a particularlyadvantageous anticoagulant medicament active principle, to the extentthat it may to this day be admitted that a preferential inhibition of anactivated factor, occuring at a stage closer to thrombin formationpractically at the intersection of said intrinsic and extrinsic routesand downstream thereof, is liable of ensuring protection against therisk of hypercoagulability, equivalent to that procured by heparincurrently used in therapeutics, without however, by reason of thisselectivity of action, resulting in the same hemorragic risks as thoseof conventional heparin. The latter is in effect adapted to inhibit notonly the Xa factor, but also other factors coming into play bothupstream and downstream of the latter, at other stages of thecoagulation routes, for example the factor IIa. It is believed that there-equilibration in vivo of the coagulation and fibrinolysis system,when the latter tends to become unbalanced under the effect of apathological cause or of an external operation, for example surgery iseasier to achieve with a medicament acting selectively on a specificfactor, the factor X, more particularly at the level of inhibition ofthe factor Xa, than with a medicament capable of acting inun-differentiated manner on several coagulation factors at once.

The invention relates also to a process for obtaining such amucopolysaccharide fraction, this process comprising:

suspending in an aqueous alcoholic medium of the water-ethanol type,having a titer comprised between about 55° and about 61° GL, preferablyof the order of 58° GL, of a substance based on heparin or heparinicconstituents whose molecular weights range notably from 2,000 to 50,000,this substance having a low content of inorganic salts, preferably lessthan 1% by weight,

separating the insoluble fraction and recovering the solution containingthe dissolved mucopolysaccharide fraction, from which it can in its turnbe separated, notably by alcoholic precipitation, from theabove-mentioned aqueous alcohol medium.

The starting material, from which the mucopolysaccharide according tothe invention may be extracted, may be constituted by a heparin ofconventional, injectable pharmaceutical quality, or by a crude heparinsuch as is obtained at the end of extraction operations for this activeprinciple from tissues or organs of mammals, notably from intestinalmucous or from lungs, for example of pork or beef. It can also beconstituted by fractions which are normally discarded (waste) in thepurification of such crude heparin, for obtaining a heparin ofinjectable quality and of higher specific activity, provided of coursethat the waste materials of lower specific activity still containheparinic constituents.

It is then possible, from raw materials of this type, substantially freefrom proteins, from nucleic acids and from inorganic salts, preferablywhen the contents by weight of the latter are less than 1%, to obtain byextraction with 55°-61° GL alcohol a mucopolysaccharide fractioncontaining constituents of low molecular weight, of which theYin-Wessler and USP titers are in a ratio of about 2 to about 5, notablyfrom 3 to 5.

It may be remarked that in using water-ethanol mixtures having more than61° GL, the extraction yield becomes practically zero. On the otherhand, the use of aqueous-alcoholic medium of a titer less than 55° GLresults in the solubilization of constituents whose presence leads tothe lowering of the ratio of the Yin-Wessler/USP titers.

It is to be noted that it is possible to proceed with additionalfractionations of the mucopolysaccharide fraction obtained at the end ofthe above-mentioned process, by various techniques, such asgel-filtration or again selective precipitation in an aqueous-alcoholicmedium of predetermined titer, in the presence of proportions alsopredetermined of an inorganic salt, such as sodium chloride.

An additional fractionation may be achieved by a supplementary stepapplied to each mucopolysaccharide fraction, previously redissolved inwater, which step consists of adding to this aqueous solution from 1 to2 volumes of ethanol and from 10 to 100 g/l of sodium chloride and ofcollecting, on the one hand, the equally active precipitate formed and,on the other hand, the content remaining dissolved in the supernatantliquor, notably by a further alcoholic precipitation, and whichconstitutes a fractionation product whose Yin-Wessler and USP titersrespectively are in a ratio still higher, of the order of 6 to 8, thanthat relating to the initial fraction, notably of the order of 3.

Mucopolysaccharide fractions having a ratio of Yin-Wessler/USP titerswhich are higher can also be obtained by gel-filtration from thefractions of the first extraction by the 55°-61° GL aqueous-alcoholmedium, after prior redissolution of the latter fractions in an aqueoussolvent, such as a 0.5M NaCl; 0.1M tris-HCl solution at pH 7.5. Such asolution may be passed through a gel of polyacrylamide and agarose, inbead form, having the tradename ULTROGEL AcA 44, whose effectivefractionating zone is situated between effective molecular weights of4,000 to 60,000 (for linear molecules).

Mucopolysaccharide fractions of the invention, which have a higherYin-Wessler/USP titer-ratios, are those in which flow after the elutionof a volume of 2.5 liters, dead volume not included (the dead volumebeing the volume of liquid contained in the column of gel, notably inthe interstitial spaces between the grains of gel), when thegel-filtration is carried out, with a flow rate of 200 ml/hour, in acolumn having a diameter of 100 mm and a height of 1 m and when theconcentration of mucopolysaccharide and the volume of solution placed onthe column have been respectively 50 mg/ml and 37.5 ml. The most activefractions are then contained in the 1.5 liters which flow subsequently.The content of the first 2.5 liters is to a great extent formed fromheparane-sulphates or heparitine-sulphates, products of higher molecularweight and of high viscosity, which do not have anticoagulant activity.

The passage from one column to another column of the same length but ofdifferent cross-section entails modification of the volume of solution(of the same concentration) to be placed on the other column, withrespect to the volume placed on the preceding column, in a ratio equalto the square of that of the cross-sections (or diameters) of thesecolumns, in order that the same fractions may be obtained in an elutionvolume from the other column itself also occuring in a ratio with thecorresponding elution volume of the preceding column substantially equalto the square of the ratio of said cross-sections.

Gel-filtrations of this type also have the additional advantage, apartfrom that which resides in the production of fractions in which theratio of the Yin-Wessler/USP titers is more favorable, of providingproducts whose solutions have low viscosity.

In this respect, it should be noted also, that the process according tothe invention of extraction of mucopolysaccharide fractions by means ofa 55°-61° GL, preferably 58° GL alcohol solution, from a commercial orpurified heparin, notably of injectable quality, still containingnotably proportions of heparane-sulphates or similar products with highmolecular weight, also constitutes in itself a process enabling thereduction in considerable proportions of the viscosity of the aqueoussolutions, which can then be formed from these heparins, thenessentially free from these mucopolysaccharide fractions.

This reduction in viscosity presents a certain advantage, having regardto the subsequent application of such heparins in anticoagulant therapy,by parenteral, notably sub-cutaneous injection.

From fractions having ratios of Yin-Wessler/USP titers of the order of 6to 8, it is possible to obtain, by additional fractionations, notably bygel filtration or the like, mucopolysaccharide fractions characterizedby ratios of Yin-Wessler/USP titers exceeding 10, notably of the orderof 13-16, and having Yin-Wessler titers higher than 130, notably 135 to160 units/mg.

It is understood that the foregoing indications of molecular weights(and which also follow, notably in the examples) are derived frommeasurements of the retention time of solutions having a predeterminedcontent of the substance studied, in experiments of gel permeationthrough a column of gel, under equally predetermined elution conditions,the logarithms of these molecular weight indications being in the samerelationship of proportionality with respect to the above-saidmeasurements of retention time, as are those of the molecular weights of4,000, 6,500, 16,000, 31,000 respectively, of polystyrene-sodiumsulphonate standards, notably those marketed by the company namedCHROMPACK (Orsay-les-Ulis, France), with respect to their respectiveretention times, measured in a system and under gel-permeationconditions which are identical.

To the extent where the treated fractions, whatever the degree ofpurification reached, are in the state of physiologically acceptablemetallic salts, such as those of sodium, they may then be converted intomixed or simple salts containing another physiologically acceptablemetal, such as calcium, by any process applicable to the salts ofheparin. Advantageously, it is possible to resort to the processdescribed in French Pat. No. 73 13580 filed Apr. 13, 1973, by Applicant.It will be recalled that this process consists essentially, starting,for example, from a sodium salt of heparin, of contacting the latterwith a different salt of another physiologically acceptable metal, forexample calcium chloride, in solution, of then proceeding with theseparation of the metallic ions unbound to the heparin (for example byalcoholic precipitation or dialysis) and, to the extent that thesubstitution ratio reached is not sufficient, of recontacting, insolution, the mixed heparin salt obtained at the end of the firstcontacting, with a further amount of another salt, notably calciumchloride, according to the desired final substitution ratio.

A further preferred MPS fraction of the present invention can further beobtained from one or the other hereabove described fractions, whichfurther fractions are characterized:

in that, in a gel-filtration operation on a gel column of polyacrylamideand agarose, in bead form, of the type marketed under the name ULTROGELAcA 44, this fraction flows through after elution of a volume of 2.5liters, dead volume not included, when the gel-filtration is conducted,at a flow rate of 200 ml/hour, in a column having a diameter of 100 mmand a height of 1 m and when the concentration of the mucopolysaccharideand the volume of the solution placed on the column have beenrespectively 50 mg/ml and 37.5 ml, the essential of this fraction beingnotably contained in the 1.5 liters of eluate which then flow through,

by a retention time of the order from 5.7 to 7.5, notably from 6.6 to7.0 minutes in a gel-permeation system on a column filled with silica ofgranulometry from 10 to 100 microns, of 250 mm in height and 9 mmdiameter, when 50 μl of a solution of 1.3 mg/ml of this fraction in a0.02M Na₂ SO₄ buffer, having been placed on this column, the elution ofsaid fraction at a flow rate of 3 ml/minute then follows.

Preferred fractions according to the present invention are characterizedmore particularly again, on the one hand, by a particular affinity withregard to antithrombin III manifested by their capacity to be fixed onthe latter, notably in a system comprising the contacting of thefractions with an antithrombin III fixed on a support, such as agarose,in an 0.2M NaCl, 0.05tris-HCl buffer at pH 7.5 and, on the other hand,by Yin-Wessler and USP titers which are in the ratio (YW/USP ratio) atleast equal to 6, the Yin-Wessler titer itself being at least equal to300 U/mg.

Preferred fractions and compounds according to the invention arecharacterized by YW/USP ratios higher than 18, with a Yin-Wessleractivity higher than 900 U/mg.

Preferably again the fractions and compounds according to the inventionare characterized by YW/USP ratios higher than 50.

The preferred compounds of the invention are characterized by YW/USPratios higher than 65 with a Yin-Wessler activity higher than 1,300U/mg.

The particular affinity of the fractions according to the invention forAntithrombin III is an essential property to be relied upon forproducing such highly enriched fractions, notably from the precedingones, which process consists of effecting selective fixation of thefurther enriched fractions or products of the present invention onantithrombin III, notably by contacting the initial fractions withimmobilized antithrombin III, particularly on a support, notablyagarose, in a buffer such as 0.2M NaCl, 0.05M tris-HCl at pH 7.5, andthen eluting the fixed fraction with a buffer of higher ionic force,sufficient to produce desorption, notably a 2M NaCl, 0.05M tris-HClbuffer.

Of course, the starting material from which the last mentioned fractionsor compounds are obtainable are not limited to the first fractionsaccording to the invention which have been defined above. Particularlythey may be obtained in any other suitable manner, notably from thecrude starting material whose nature has been recalled above and fromwhich said first fractions themselves were obtained.

The invention relates more particularly also to the substantiallyhomogeneous compounds and in state of substantial purity which appear toconstitute the essential active principle of the preceding fractions.

These compounds are characterized by nuclear magnetic resonance spectra(NMR) carried out under the conditions indicated below and which are thesubject of FIGS. 11, 12, 14 and 15.

Referring more particularly to the NMR spectrum of the compoundsaccording to the invention for the proton (¹ H) carried out on solutionsof these compounds dissolved in deuteriated water at 35° C. with aradiation of 270 megahertz (MHz) there are observed as characteristicelement of the spectrum, resonance signals which, for chemicaldisplacement of the order of 4.8 and 5.2 ppm, are substantially weakerthan the resonance signal which is also observed for a chemicaldisplacement of the order of 5.4 ppm (reference for the measurement ofthe displacements: TSP (sodium 3-trimethylsilyl propionate 2,2,3,3-d₄)).

The signals observed at the level of the chemical displacements of 5.4;5.2 and 4.8 ppm correspond to the signals which, in the case of aconventional heparin studied by NMR under the same conditions, arerespectively characteristic of the:

anomer proton, in the 1 position, of the glucosamine N-sulphated unitsof heparin (signal G₁);

anomer proton in the 1 position, of 2-O-sulphated iduronic acid units(signal I₁) and

proton in the 5 position of 2-O-sulphated iduronic acid units (signalI₅).

In conventional heparins, the (G₁), (I₁) and (I₅) signals have all threeintensities of the same order of magnitude.

For convenience of language, reference will also be made below, even asregards the fractions or compounds according to the invention, to thesignals (G₁), (I₁) and (I₅), to denote the signals observed inrelationship with the corresponding chemical displacements (whether forthe proton or for ¹³ C).

This equivalence of language will also extend to the NMR spectrumproduced under different conditions and with different references.

Referring more particularly to the NMR spectrum of the compoundsaccording to the invention for carbon 13 (¹³ C), effected on solutionsof these compounds dissolved in deuteriated water with a radiation of 20MHz, there are observed as characteristic elements of the spectrum(reference for the measurement of the TMS (tetramethylsilane)):

the absence practically of the resonance signal characteristic of thepresence of OH groups on the primary carbon (in the 6 position) of theglucosamine units contained in the mucopolysaccharide fractions of theinvention,

additional signals, in the region of the (l₁) and (G₁) signals, inregions corresponding to chemical displacements of the order of 100 ppm,

an additional (G₂) signal close to the G₂ N-sulphate signal in the 60ppm region,

the presence of a resonance signal in the 75 ppm region (to whichnormally substantially no resonance signal corresponds in the NMRspectra obtained under similar conditions with a conventional heparin),(the indications of chemical displacements indicated above are estimatedwith respect to the CH₃ of the N-acetyl glucosamine groups contained inthe MPS according to the invention (25 ppm region in the spectra of thedrawings)).

The homogeneous compounds according to the invention, in the practicallypurified state, which all have the characteristics which have beendescribed already above, as regards their USP and Yin-Wessler activitiesand their specific affinities for antithrombin III, are alsocharacterized in that they are formed by a homogeneous oligosaccharidehaving again the following additional characteristics:

it comprises from 8 to 12, notably 10 monosaccharide units;

all the primary positions of the glucosamine units of thisoligosaccharide are sulphated;

this oligosaccharide comprises one N-acetyl glucosamine unit for two2-O-sulphate iduronic acid units and for two N-sulphate-glucosamineunits, the other saccharides being of a different nature and includingseparate substitutents.

The molecular weights of certain at least of the oligosaccharidesaccording to the invention are situated in a range from about 2,000 toabout 3,000, notably from about 2,500 where decasaccharides areconcerned.

The invention relates also to polysaccharides having the above-indicatedgeneral properties, as regards more particularly the USP and Yin-Wessleractivities, on the one hand, and the affinity for antithrombin III, onthe other hand, these fractions having a higher molecular weight, butalso containing in their structure an oligosaccharide part having theabove mentioned structure.

Other characteristics of the invention will appear also in the course ofthe description which follows of preferred examples of the practising ofthe invention, notably with reference to the drawings in which:

FIG. 1 shows a characteristic elution diagram of a preferredmucopolysaccharide fraction, according to the invention.

FIG. 1a is the NMR spectrum of a mucopolysaccharidic fraction accordingto the invention.

FIGS. 2 to 7 show the comparative biological properties ofmucopolysaccharide fractions according to the invention and of aconventional heparin with high anticoagulant activity (in USP titer).

FIG. 8 is a diagrammatic elution diagram of a fraction according to theinvention, on the practising of the process, also according to theinvention, of selective separation of said fraction from a fraction alsocontaining other constituents;

FIG. 9 is representative of an elution diagram characteristic of anotherpreferred mucopolysaccharide fraction, according to the invention;

FIG. 10 is the NMR spectrum of a conventional heparin for the ¹ Hproton;

FIGS. 11 and 12 are NMR spectra for the ¹ H proton of differentfractions according to the invention;

FIG. 13 is an NMR spectrum for the 13 carbon of a conventional heparinused as a comparison;

FIG. 14 is an NMR spectrum for the carbon 13 of a fraction according tothe invention, and

FIG. 15 is an enlargement of a part of the NMR spectrum of FIG. 14.

EXAMPLE 1

The raw material was constituted by 100 g of an injectable heparinhaving a titer of 170 IU/mg (USP units).

To this 100 g of heparin, 2,500 ml of 58° GL alcohol are added. Aftervery vigorous stirring for 15 minutes, vigorous stirring is continuedfor 15 hours. The suspension is then centrifuged at 7,000 rpm for 1 hourand the supernatant liquor is recovered: 2,400 ml.

To this supernatant liquor 80 ml of saturated sodium chloride solutionand then 2,400 ml of 100° GL alcohol are then added.

The precipitated product is recovered, washed with alcohol and dried. Itweighs 2.1 g.

It will be called hereafter LMF (low molecular weight fraction). Resultsregarding the physical and chemical properties of this fraction aregiven herebelow.

Results obtained with commercial heparin, i.e. unfractionated heparin(designated U H) are given for purpose of comparison.

I--Physical and chemical properties (1°) molecular weight (HPLC method)

L M F: 4,000-8,000 U H: 4,000-30,000

(2°) analytical results

Sulfate, N-acetyl glucosamine, glucuronic acid and iduronic acid contentof LMF and UH were determined. The results are given in Table I.

                  TABLE 1                                                         ______________________________________                                                         LMF   UH                                                     ______________________________________                                        SULFATE            26%     34%                                                N--ACETYL           7%      3%                                                GLUCOSAMINE                                                                   GLUCURONIC         11%      6%                                                ACID                                                                          IDURONIC           24%     24%                                                ACID                                                                          ______________________________________                                    

It appears from these results that the LMF has a content in N-acetylglucosamine and acid glucuronic moieties higher than heparin. Thecontent in iduronic acid units appears to be similar. The sulfatecontent is somewhat less than in heparin.

(3°) N M R spectrum

The ¹³ C spectrum was recorded oon solutions of this fraction dissolvedin deuteriated water with a radiation of 20 MHz (see FIG. 1a).

The typical signals observed are the followings:

23.4 ppm: CH₃ of NH-acetyl group

54.7 ppm: carbons in position 2 of the acetylated glucosamine moieties

59.2 and 58.8 ppm: carbons in position 2 of the sulfate glucosaminemoieties (splitting).

61.7 ppm: carbons in position 6 with --OH groups

67.4 ppm: carbons in position 6 with --O-sulfated groups

98.3** ppm: carbons in position 1

100.00 ppm**: carbons in position 1 of the sulfated iduronic acid units

≃103 ppm: carbons in position 1 of the glucuronic acid units.

(4°) Electrophoretic analysis

The electrophorogram (cellulose acetate, HCl) consists of two bands,both in the heparin region. EXAMPLE II

The raw material used was derived from sub-fractions such as areobtained in the purification of commercial heparin, for the productionof injectable heparin. It is obtained notably in part from thesupernatant liquor obtained by the addition of 0.6 to 0.7 volume of 100°GL alcohol to an aqueous solution of heparin containing 10 to 20 g perliter of sodium chloride, the precipitated purified heparin then beingrecovered for purification. The raw material used here also containedvarious heparin purification residues, notably those obtained byalcoholic precipitations, for freeing injectable heparin from traces ofinorganic salts.

To 10 kg of this raw material is added 30 volumes of 58° GL (300 liters)of alcohol. The suspension is subjected to vigorous dispersion andagitation for 15 minutes, the stirring being further maintainedenergetically for 12 hours. It is then left to stand for 48 hours, inorder to produce precipitation of the non-solubilized raw material. Theslightly cloudy supernatant liquor is taken up again and clarified bycentrifugation.

To the supernatant liquor (volume of 280 liters) is added 10 liters of asaturated solution of sodium chloride, and then 1 volume (280 liters) of100° GL alcohol. The precipitate obtained, which contains themucopolysaccharide fraction, is washed with 100° GL alcohol, and thendried.

660 g of a fraction are obtained whose Yin-Wessler and USP titersrespectively are already in a ratio higher than 2 (fractionP194HH.sub.(A)).

A supplementary fractionation is then made from this fraction, bydissolving the 660 g fraction in 13,200 ml of water.

To the solution formed is added 264 g of sodium chloride, and then 1.5volumes of 100° GL alcohol (19.8 liters). The precipitated product iscollected, washed with alcohol, then dried. 640 g of the P194HH.sub.(C)fraction are obtained, having the following characteristics:

USP titer: 31 IU/mg,

Yin-Wessler titer: 100 U/mg.

The supernatant liquor contained also active mucopolysaccharidefractions (their recovery is described in Example IV).

The P194HH.sub.(C) fraction contains also a relatively large amount ofsubstances of high molecular weight mainly heparitine-sulphates, withoutanticoagulant activity, both in the USP test and in the Yin-Wesslertest.

After redissolving in a 0.5M NaCl, 0.1M tris-HCl buffer at pH 7.5, inthe proportion of 50 mg/ml, a gel-filtration follows of a volume of 150ml of the solution on AcA44, in a column of diameter of 215 mm, of 1meter height, with a flow rate of 800 ml/hour. The high molecular weightsubstances, of which the major portion is heparitine-sulphates passes inthe 10 first liters of eluted solution, dead volume not included.

A mucopolysaccharide fraction with higher Yin-Wessler titer, with aratio of the Yin-Wessler/USP titers of the order of 4 to 8, can beobtained from the next 6 liters of eluate.

EXAMPLE III

This example describes a modification of the processing of theP194HH.sub.(C) fraction of Example II. After it being dissolved again ina 0.5M NaCl, 0.1M tris-HCl buffer at pH 7.5, in a proportion of 50mg/ml, it is subjected to a gel-filtration on ULTROGEL AcA 44, in acolumn of 10 cm diameter, 100 cm height. The elution flow rate was 200ml/hour.

The eluate as collected in successive fractions of 50 ml. Themucopolysaccharide content of each fraction was evaluated as follows: to1 ml of the fraction was added 2 ml of 100° GL alcohol. After standingfor 2 minutes, the turbidity of te mixture was measured at 660nanometers, on a spectrometer (optical density measurements). Thisturbidity was directly proportional to the mucopoly-saccharide contentof the tested solution.

The C10 fraction, contained in the last third of the fourth liter ofeluate, dead volume not included, was collected. The ratio of theYin-Wessler/USP titers of the C10 fraction was 50/6.

EXAMPLE IV

The final supernatant liquor of Example II is itself supplemented with19.8 liters of 100° GL alcohol and the suspension formed allowed tostand for 24 hours.

The precipitate formed was collected, washed with 100° GL alcohol anddried. 6 g were obtained of a fraction called P194HH.sub.(P) having thefollowing characteristics:

USP titer: 7 IU/mg,

Yin-Wessler titer: 46 U/mg.

EXAMPLE V

The P194HH.sub.(P) fraction was again dissolved in a 0.5M tris-HCl, 30g/l NaCl buffer at pH 7.5, in the proportion of 50 mg/ml.

The solution was subjected to gel filtration on an ULTROGEL AcA 44column (Pharmacia K 100/100, volume: 7 liters; height 100 cm; diameter10 cm) with a flow rate of 200 ml/hour.

The elution diagram obtained is shown diagrammatically in FIG. 1,showing the variations in the content of material (optical density DOmeasured at 660 nanometers) as a function of the eluted volume, inliters (1).

There was collected, after passage of a volume of liquid correspondingto the dead volume of the column, successive fractions K, J, I, G, F, E,D, C, B and A, whose volumes are indicated by the length of thecorresponding abscissae segments of FIG. 1.

Each of these fractions possess analytical characteristics which areshown in the following table.

                  TABLE I                                                         ______________________________________                                        Characteristics of the Fractions Obtained                                      No.Fraction                                                                          Weight (mg)                                                                            USP titer (IU)                                                                          Anti-Xa titer(U)                                                                       ##STR1##                                  ______________________________________                                        A       120      3.7       44.4    12                                         B       120      4.5       72      16                                         C       250      6         54       9                                         D       150      9         135     15                                         E       300      9         144     16                                         F       400      11        143     13                                         G       300      11.5      161     14                                         H       200      13        143     11                                         I       50       13        91       7                                         J       200      7         14       2                                         K       3500     0         0       /                                          ______________________________________                                    

It is observed that the fractions can be grouped into four types:

(a) The Fractions A, B, C whose elution volumes corespond, in theabove-described operational procedure, essentially to the fourth litereluted, whose USP titers are less than 10 and Yin-Wessler titers lessthan 80; their molecular weights are at the most of the order of 4,000;

(b) The fractions D, E, F, G, H, whose USP titers are less than 10 andYin-Wessler titers very high: 135 to 161 units; these fractions alsohave the most favorable Yin-Wessler/USP titer ratios, from 13 to 16;they are essentially contained in the third liter of eluate; theirmolecular weights are of the order of 4,000 to 10,000, notably from4,000 to 8,000;

(c) The fractions I and J, whose ratios of Yin-Wessler/USP titers tendto become unfavorable, and which are probably already contaminated withthe K fraction below and

(d) The K fraction, containing again essentially heparane-sulphatesdevoid of anticoagulant activity.

In Table II are displayed the molecular weights of certain of thefractions estimated according to the retention time measured ingel-permeation, by reference to those of the abovesaidpolystyrene-sulphonates of known molecular weight. The fraction F ischaracterized by a main peak corresponding to a retention time of 6.6minutes and by a shoulder corresponding to a retention time of 6.1minutes, which testifies to the presence of a constituent whosemolecular weight is situated towards 7,200 in the reference systemconcerned.

The measurements were done by gel-permeation (by means of aSPECTRAPHYSICS 3500 chromatograph), on columns (250×9 mm) filled withsilica of granulometry 10-100 microns, notably those marketed under thename LICHROPHOSPHER, of solutions of these fractions in a 0.02M Na₂ SO₄buffer in the proportion of 1.3 mg of mucopolysaccharide material/ml(volume initially deposited on the column: 50 μl) and with an elutionflow rate of 3 ml/minute. The detection of the material was done by UVspectrophotometry (200 mμ).

                  TABLE II                                                        ______________________________________                                                                 Molecular weights                                                 Retention time                                                                            relative to                                          Product      (minutes)   polystyrenes                                         ______________________________________                                        P194HH.sub.(A)                                                                             7.0         2,600                                                P194HH.sub. (B)                                                                            6.9         2,900                                                P194HH.sub. (C)                                                                            6.8         3,300                                                P194HH.sub. (F)                                                                            6.6         4,100                                                P194HH.sub. (F)                                                                            6.1*         7,200*                                              polystyrene- 6.6         4,000                                                sulphonate (1)                                                                polystyrene- 6.2         6,500                                                sulphonate (2)                                                                polystyrene- 5.4         16,000                                               sulphonate (3)                                                                polystyrene- 4.7         31,000                                               sulphonate (4)                                                                ______________________________________                                         *shoulder                                                                

EXAMPLE VI Raw Material

It is formed of byproducts derived from the manufacture of injectablecalcium heparinate from crude heparins, such as those extracted fromanimal tissues (notably intestinal mucous or beef or pork lungs).follows had the following characteristics:

Weight: 252 kg USP titer: 82 IU/mg Yin-Wessler titer: 100 IU/mg.

The processing steps described below were then resorted to.

Extraction with 58° GL alcohol

The 252 kg of raw material were dispersed in 6,000 liters of 58° GLalcohol with vigorous stirring.

The insoluble phase was separated by decantation and centrifugation.

The soluble fraction was recovered by the addition of NaCl and 100° GLalcohol.

There was obtained:

Insoluble fraction: 230 kg recycled in manufacture,

Soluble fraction: 20.6 kg (USP titer=21 IU/mg Yin-Wessler titer=90IU/mg).

The extraction of the fraction of low molecular weight from the fraction

The fraction soluble in 58° GL alcohol was dissolved in 512 liters ofwater (20 volumes).

10.24 kg of NaCl were added and then 1.5 volumes of 100° GL alcohol,namely 768 liters. The precipitated fraction was collected, dehydratedwith alcohol and dried.

Weight: 19 kg USP titer: 22 IU/mg Yin-Wessler titer: 89 U/mg.

This fraction was put aside and subsequently purified and converted intoan injectable calcium salt.

The supernatant liquor from the precipitation at 1.5 volumes wassupplemented with 1.5 volumes of alcohol, namely 768 liters. Thefraction precipitated was collected, dehydrated with alcohol and dried.

Weight: 700 grams USP titer: 6 IU/mg Yin-Wessler titer: 40 U/mg.

This fraction of 700 grams was a mixture of low molecular weight MPS,little sulphated high molecular weight MPS and more or less degradednucleic acids.

Removal of the nucleic acids from the low molecular weight fraction

The major part of the nucleic acids was removed by precipitation withmanganese chloride, in the following manner:

The fraction of 700 grams was dissolved in 7 liters of water. 1 liter of10% MnCl₂ was added with stirring. The considerable precipitate formed(constituted by the insoluble manganese salts of the RNA and DNA) wasremoved by centrifugation. The MPS was recovered from the clearsupernatant liquor by precipitation with alcohol.

Weight: 480 grams USP titer: 8 IU/mg, Yin-Wessler titer: 54 U/mg.

Isolation of the fractions of very low molecular weights bygel-filtration

The very low molecular weights were separated by gel-filtration onULTROGEL AcA 44.

A column of 200 mm diameter and 1 m height enabled 25 grams to betreated.

The elution diagram is of the type shown in FIG. 8.

Three fractions (numbered from (1) to (3)) were collected. They had thefollowing characteristics (for 25 grams applied at the start):

(1) weight: 16 grams USP titer: 12 IU/mg Y.W. titer: 30 U/mg,

(2) weight: 7 grams USP titer: 6.5 IU/mg Y.W. titer: 70 U/mg,

(3) weight: 2 grams USP titer: 2.1 IU/mg Y.W. titer: 60 U/mg.

Chromatography on insolubilized antithrombin III

The preceding fraction (3) was subjected to chromatography onantithrombin III fixed on agarose.

A column of 100 ml used at present enabled 700 mg of the fraction (3) tobe treated.

The absorption was effected in an 0.2M NaCl, 0.05M tris-HCL buffer at pH7.5.

The elution was carried out by a 2M NaCl, 0.05M tris-HCL buffer.

The unfixed portion (600 to 650 mg) had a USP titer close to 1 to 2IU/mg and a Yin-Wessler titer from 10 to 20 U/mg.

The fixed portion (10 to 30 mg) had a USP titer from 10 to 20 IU/mg anda Yin-Wessler titer from 1000 to 1400 U/mg.

EXAMPLE VII

The fractions A, B and C for Example V hereabove were pooled into asingle fraction which was then subjected to an additional fractionationby selective fixation on an agarose-antithrombin III column, under theconditions defined in Example VI.

The fixed fraction was eluted. The fraction named below P194HPA wasobtained. It possessed a Yin-Wessler titer of 310 U/mg and a USP titerof 40 IU/mg.

In the same way the above-mentioned fractions E and F of Example V werepooled. The separation procedure of the most active fractions by thetechnique of fixation-elution defined above, by means of theagarose-antithrombin III column, was resorted to again. Finally therewas obtained a fraction P194HHPF having a Yin-Wessler titer of 900 U/mgand a USP titer of 82 IU/mg.

The said fractions P194HHPA and P194HHPF were subjected to NMR analysis,for the ¹ H proton. The same was done with a conventional heparin(7021HH).

The analysis was carried out on each of the said products, previouslydissolved in deuteriated water in the proportion of 14 to 62 mg/0.35 ml,with a BRUKER apparatus, 270 MHz, equipped with a FOURIER transformsystem and enabling the storage of accumulating spectra. The chemicaldisplacements were measured with reference to TSP, as indicated above.

FIG. 10 is representative of the NMR spectrum obtained with conventionalheparin. FIGS. 11 and 12 are in the same way representative of the NMRspectra of fractions P 194HHPF and P 194HHPA.

It is noted, by comparison of the NMR spectra, that the (I₁) and (I₅)signals of the fractions according to the invention are distinctly lessintense than the signal (G₁), whereas these signals are substantially ofthe same intensity in the heparin reference spectrum.

EXAMPLE VIII

By applying the techniques described in Examples VI and VII to otherstarting materials, there were obtained similarly fractions:

    ______________________________________                                        P 219 HH:    USP titer      = 14 IU/mg                                                     Yin-Wessler titer                                                                            = 1350 U/mg                                       P 225 HH:    USP titer      = 17 IU/mg                                                     Yin-Wessler titer                                                                            = 1320 U/mg                                       P 231 HH:    USP titer      = 16.2 IU/mg                                                   Yin-Wessler titer                                                                            = 1400 U/mg                                       P 194 HH A:  USP titer      = 82 IU/mg                                                     Yin-Wessler titer                                                                            = 900 U/mg                                        P 242 HH A:  USP titer      = 16 IU/mg                                                     Yin-Wessler titer                                                                            = 1800 U/mg                                       P 255 HH A:  USP titer      = 36 IU/mg                                                     Yin-Wessler titer                                                                            = 2145 U/mg                                       ______________________________________                                    

The P 242HHA fraction was subjected to NMR analysis, for the 13 (¹³ C)carbon (FIGS. 14 and 15). The same was done with conventional heparin ofreference 7071HH (FIG. 13). The analysis was carried out on each of thefractions (in solution in deuteriated water in the proportion of 100 mgin 1 ml of D₂ O with a VARIAN CFT-20, 20 MHz apparatus, equipped with aFOURIER transform system (reference for the measurement of chemicaldisplacements: TMS).

There is observed:

the absence practically of a resonance signal characteristic at thepresence of OH groups on the primary carbon (in the 6 position of theglucosamine units contained in the mucopolysaccharide fractions of theinvention),

additional signals (not contained in the NMR spectrum of the referenceheparin in the region of the (l₁) and (G₁) signals, in regionscorresponding to chemical displacements of the order of 100 ppm,

a supplementary signal in the 60 ppm region close to the (G₂) signal,

the presence of a resonance signal in the 75 ppm region (to whichnormally no resonance signal corresponds in the NMR spectra producedunder similar conditions with conventional heparin), (the indications ofchemical displacements indicated above are evaluated with respect to theCH₃ of the N-acetyl glucosamine groups contained in the MPS according tothe invention (region of 25 ppm in the spectra of FIGS. 14 and 15).

Signals particular to the fractions or compounds according to theinvention are marked with an asterisk in FIGS. 14 and 15.

FIG. 15 also includes the CI integration curve, which enables it to beobserved that:

the compound studied was homogeneous, hence practically pure,

it has the characteristics of a decasaccharide,

it includes one N-acetyl glucosamine unit, for two units of 2-O-sulphateiduronic acid and for two N-sulphate-glucosamine units.

The invention hence enables the preparation of mucopolysaccharidefractions with high anti-Xa activity and having with respect to the Xafactor a remarkable selectivity in the framework of successive enzymaticreactions which characterize the coagulation process.

This remarkable activity and selectivity are also illustrated by theresults of the pharmacological tests described below, which were carriedout with the P188CH fraction, obtained after the conversion of theP194HHC fraction of Example II, which was in the sodium salt form, intothe calcium salt form, by the above-recalled process.

These results are illustrated by the curves of FIGS. 2 to 7, which areall intended to show the comparative anticoagulant effects of themucopolysaccharide fraction of the invention, on the one hand, and of aconventional heparin (170 USP units/mg), on the other hand.

The curves of FIGS. 2 to 5 are illustrative of the variation observed invitro of the coagulation times induced in human blood plasmas byincreasing doses of a conventional heparin on the one hand, and of theP188CH fraction, on the other hand (the tests corresponding to FIGS. 4and 5 having been carried out on plasmas free of platelets andconsequently impoverished in factor XI).

FIGS. 6 and 7 relate to the comparative results obtained in vivo in therabbit, with the same P188CH fraction (FIG. 6) and the reference heparin(FIG. 7) (average of the results obtained on groups of five rabbits).Each of the rabbits had received 500 Yin-Wessler units per kg of thecomposition to be tested.

Concerning firstly FIGS. 2 to 5, they show the variations of the times(in seconds):

of thrombin (FIG. 2),

of cephalin-kaolin (FIG. 3),

of coagulation in the presence of concentrated thromboplastin (FIG. 4)and of diluted thromboplastin (FIG. 5), induced respectively by thepreparations studied, namely the mucopolysaccharide fraction (curves a₁,a₂, a₃ and a₄) and the reference heparin (curves b₁, b₂, b₃ and b₄) as afunction of the respective doses used, all expressed in USP units/ml.

The thrombin time and the cephalin-kaolin time both constitute types ofmeasurement reflecting rather the action of the preparations studiedrespectively on the inhibition of the activated factor II and theoverall coagulation. The curves of FIGS. 2 and 3 clearly show in thisrespect that the mucopolysaccharide fraction according to the inventionexerts a distinctly lesser effect than that of the heparin of comparisonon the inhibition of the inactivation of a prothrombin and at the levelof the overall coagulation. In contrast, FIGS. 4 and 5, which arerepresentative of the phenomena more directly connected with thesequence of enzymatic reactions characteristic of extrinsic coagulation(notably in the relative absence of the factor IIa) show a distinctadvantage of the mucopolysaccharide fraction of the invention withrespect to the reference heparin. The MPS fraction causes under theseconditions a slower coagulation of the blood specimen.

In FIG. 6, there are shown the variations of the activities measured ina rabbit which has received 500 Yin-Wessler units of amucopolysaccharide fraction of the invention, as a function of time,expressed in hours. To evaluate these activities, recourse is had to thevariation of the Yin-Wessler titers (curve YW₅) and the cephalin-kaolintiters (curve CKT₅) (IU/ml plasma) as a function of time in hours (H).

The same measurements were carried out with the reference heparin. Thecorresponding variations of the activities studied are illustrated bycurves YW₆ and CKT₆ of FIG. 7.

If FIG. 6 is examined, it is observed that the administration of 500Yin-Wessler units of the mucopolysaccharide according to the inventioncauses a considerable anti-Xa activity, compared with the overallcoagulability effect, expressed in CKT units, which remains relativelylow. It is noted, for example, that at the second hour, the Yin-Wessleractivity is 0.85 U/ml, whilst the CKT activity is only 0.15 IU/ml. Tothe contrary 500 Yin-Wessler units/ml of reference heparin induce aneffect expressed by the CKT titers, which is distinctly greater relativeto the anti-Xa activity measurable by the Yin-Wessler titer. Inparticular, it is noted that at the second hour, the anti-Xa activitycorresponds to 0.55 U/ml, and that the overall anticoagulant activity,CKT, is of 0.38 IU/ml. The difference between the two titers is hencemuch smaller than in the case of the mucopolysaccharide according to theinvention. The ratio of the Yin-Wessler titer to the CKT titer hencepasses from a value less than 2 for the reference heparin to a valuegreater than 5 for the mucopolysaccharide fraction of the invention.

In vitro and in vivo tests are hence both in the sence of a distinctlymore selective action of the mucopolysaccharide fraction of theinvention, notably at the level of inhibition of the Xa factor, thanthat of the reference heparin.

The mucopolysaccharide fractions according to the invention are free oftoxicity. The administration of 10,000 U/kg (Yin-Wessler titer) of anyof the fractions according to the invention causes in the rabbit neitherany toxic reaction nor any pyrogenic effect in the pyrogenicity test inthe rabbit according to the French Pharmacopoea.

The invention hence relates more particularly to mucopolysaccharidefractions of the type which have been described, having notably anactivity of at least 40, preferably at least 50, and even moreadvantageously again of at least 100 U/mg (Yin-Wessler titer). Fractionscontaining more than 300, particularly more than 900 U/mg (Yin-Wesslertiter) are even more preferred. It relates also to pharmaceuticalpreparations, having similar activities, devoid of pyrogenic substances,and in association with pharmaceutical excipients. It relates inparticular to the injectable, sterile, concentrated solutions of thesefractions, useful in therapeutics, for the control of blood coagulation,which solutions contain from 1,000 to 100,000 U (Yin-Wessler)/ml of themucopolysaccharide fraction, preferably from 5,000 to 50,000, forexample 25,000 U/ml, when these solutions are intended for sub-cutaneousinjection or containing again, for example, from 500 to 10,000, forexample 5,000 U units/ml of the mucopolysaccharide fraction, when theyare intended for intravenous injection or for perfusion.

The mucopolysaccharide fraction according to the invention isadvantageously in the form of a salt of at least one physiologicallyacceptable metal, such as sodium and/or calcium. Advantageously, thesepharmaceutical proportions are presented in the form of syringes usableonly once, ready for use at any suitable time.

The compositions according to the invention are particularly adapted tothe control (preventive or curative) of the blood coagulation in man oranimal, notably in those cases where the host is subjected to risks ofhypercoagulability, more particularly those resulting from disturbanceof the abovesaid extrinsic phase, for example, as a consequence of therelease by the organism of thromboplastin, for example, of tissularthromboplastin (surgical operations, atheromatous processes, tumordevelopment, disturbances of the coagulation mechanisms by bacterial orenzymatic activators, etc.). For the sole purpose of illustrating theinvention, and without there being discoverable therein cause forlimiting the protection of the invention, there will be indicated below,by way of example, a posology capable of being used in man: it comprisesfor example, the administration to the patient of 1,000 to 25,000 U bythe sub-cutaneous route, 2 to 3 times daily, according to the level ofhypercoagulation risk or the thrombotic condition of the patient, orfrom 1,000 to 25,000 U per 24 hours by the intravenous route, indiscontinuous administration at regular intervals or continuously byperfusion, or again from 1,000 to 25,000 U (three times weekly) by theintramuscular route (titers expressed in Yin-Wessler U). The dosesshould naturally, be adjusted in each patient according to the resultsof previously effected blood analyses, the nature of the disorder fromwhich the patient is suffering and, generally, his state of health, asis well known.

The invention again also relates to the application of themucopolysaccharides according to the invention to the constitution ofbiological reactant usable in laboratory, notably as a comparisonreference for the study of other substances of which the anticoagulantactivity is to be tested, notably at the level of inhibition of thefactor Xa.

As is self-evident and as emerges already from the foregoing, theinvention is in no way limited to those of its types of application andembodiments which have been more especially envisaged; it encompasses onthe contrary all modifications, in particular those in which theaqueous-alcoholic extraction medium defined above is formed by a mixtureof water and an alcohol other than ethanol, for example an aliphatic oraromatic alcohol, preferably cyclic or acyclic saturated aliphaticalcohol, such as primary alcohols including 1 to 6 carbon atoms, itbeing of course understood that there should be determined in each case,by simple routine operations, the proportions of water/alcohol of themedium which lead to an extraction of a mucopolysaccharide fractionequivalent to that which is obtained with a 55°-60° GL water-ethanolmixture.

Finally it is to be noted that all definitions set forth in the claimsthat follow are whenever appropriate also part of the presentdisclosure.

We claim:
 1. Heparinic mucopolysaccharide fractions which have improvedantithrombotic activity in vivo (measured in terms of activity ofanti-Xa per milligram), which are more selective with respect to anti-Xaactivity than that of heparin and have a lower whole anticoagulationactivity than heparin (measured in USP units per milligram), whichheparinic mucopolysaccharide fractions have (1) a molecular weight inthe range of about 2,000 to 10,000 daltons, (2) are soluble inwater-alcohol having a titer of 55°-61° GL, (3) are insoluble inalcohol, (4) have a ratio of anti-Xa titer to USP titer of at least 3,wherein in said heparinic mucopolysaccharides, fractions comprise, (5)glucosamine units which are sulfated in the primary position, (6) oneN-acetyl glucosamine unit for two 2-O-sulfate iduronic acid units andfor two N-sulfate-glucosamine units, the carbon-13 NMR spectrum in H₂ O(at a radiation of 20 MHz) having the following resonance signals (inthe stated regions) indicating the presence of the stated atoms:

    ______________________________________                                        23.4 ppm:      CH.sub.3 of NH--acetyl group,                                  54.7 ppm:      carbons in position 2 of the                                                  acetylated glucosamine moieties,                               59.2 and 58.8 ppm:                                                                           carbons in position 2 of the                                                  sulfate glucosamine moieties                                                  (splitting),                                                   61.7 ppm:      carbons in position 6 with --OH                                               groups,                                                        67.4 ppm:      carbons in position 6 with                                                    --O--sulfated groups,                                          98.3.sup.x x ppm:                                                                            carbons in position 1,                                         100.00 ppm.sup.xx :                                                                          carbons in position 1 of the                                                  sulfated iduronic acid units, and                              103 ppm:       carbons in position 1 of the                                                  glucuronic acid units,                                         ______________________________________                                    

and the physiologically acceptable salts thereof.
 2. Themucopolysaccharides of claim 1, which have a carbon-13 NMR spectrum asshown in FIG. 1a.
 3. The mucopolysaccharides of claim 1, the carbon-13NMR spectrum of which exhibits glucosamine units, the primary carbons inthe 6-position being free of hydroxyl group and exhibiting resonancesignals in the region corresponding to chemical displacements in the 100ppm region (as shown by stars in FIG. 14).
 4. The mucopolysaccharides ofclaim 2, wherein the carbon-13 NMR spectrum exhibits another resonancesignal in the 75 ppm region.
 5. The mucopolysaccharides of claim 4,which proton NMR spectrum exhibits resonance signals in the 4.8, 5.2 and5.4 ppm regions,, which signals in the 4.8 and 5.2 ppm regions areweaker than that in the 5.4 ppm region.
 6. The mucopolysaccharides ofclaims 3, wherein the carbon-13 NMR spectrum exhibits a supplementarysignal in the 60 ppm region adjoining the G₂ designated signal (as shownin FIGS. 14 and 15).
 7. The mucopolysaccharides of claim 1, as shown byone of the NMR spectra of FIGS. 11, 12, 14 or
 15. 8. Themucopolysaccharides of claim 1, which is selectively fixable onantithrombin III.
 9. The mucopolysaccharides of claim 1, which have aUSP titer of about 45 units per mg, an anti-Xa titer of about 160 unitsper mg and a ratio of anti-Xa titer to USP titer of about 3.55.
 10. Themucopolysaccharides of claim 1, wherein the USP titer is less than about10 units per mg and the anti-Xa titer is about 161 units per mg.
 11. Themucopolysaccharides of claim 1, wherein the ratio of anti-Xa titer toUSP titer is at least 6, and the anti-Xa titer is at least 300 units/mg.12. The mucopolysaccharides of claim 11, wherein the ratio of anti-Xatiter to USP titer is at least
 10. 13. The mucopolysaccharides of claim12, wherein the ratio of anti-Xa titer to USP titer is at least
 50. 14.The mucopolysaccharides of claim 11, wherein the ratio of anti-Xa titerto USP titer is at least
 130. 15. The mucopolysaccharides of claim 11,wherein the anti-Xa titer is not less than about 50 units per mg. 16.The mucopolysaccharides of claim 11, wherein the ratio of anti-Xa titerto USP titer is higher than 18 and the anti-Xa titer is at least 900units per mg.
 17. The mucopolysaccharides of claim 13, wherein the ratioof anti-Xa titer to USP titer is higher than 65 and the anti-Xa titer isat least 1300 units per mg.
 18. The mucopolysaccharides of claim 1,wherein the USP titer does not exceed about 13 units per mg, the anti-Xatiter is in the range of about 135 to about 160 units per mg, the ratioof anti-Xa units to USP units is in the range of 13 to 16 and themolecular range is from about 4,000 to about 8,000 daltons.
 19. Themucopolysaccharides of claim 1, wherein the USP titer does not exceedabout 6 units per mg, the anti-Xa titer is not less than about 44 unitsper mg, the ratio of anti-Xa to USP titers is over about 9 and themolecular weight is in the range of about 4,000 to 8,000 daltons. 20.The mucopolysaccharides of claim 1, wherein the salts are selected fromthe group consisting of sodium and calcium.
 21. A therapeuticcomposition which comprises a therapeutically acceptable carrier and inan antithrombotic effective amount, the heparinic mucopolysaccharidefractions of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or
 20. 22. The therapeutic composition of claim 21, whichhas an anti-Xa titer higher than about 100 units per mg.
 23. Thetherapeutic composition of claim 21, which is a solution of themucopolysaccharides in a concentration of about 1,000 to 100,000Yin-Wessler units per ml.
 24. The therapeutic composition of claim 23,which is a solution of the mucopolysaccharides in a concentration ofabout 5,000 to about 50,000 units per ml.
 25. A therapeutic method forcontrolling thrombosis in a patient which comprises administering to thepatient in a therapeutically antithrombotic effective amount, thecomposition of claims 21, 22, 23 or 24 and controlling thrombosis byselectively inhibiting the coagulation factor Xa.
 26. The therapeuticmethod of claim 25 wherein the administration of the composition is byinjection or infusion.
 27. A process for obtaining heparinicmucopolysaccharides which have improved antithrombotic activity in vivoand inhibition of the Xa-factor (measured in terms of anti-Xa activity)more selective than that of heparin and a lower whole anticoagulationactivity than heparin (measured in USP units), which mucopolysaccharideshave a molecular weight in the range of about 2,000 to 10,000 daltons, aratio of anti-Xa to USP titers of at least 3, which process comprisesmixing heparin mucopolysaccharides having a molecular weight in therange of about 2,000 to 50,000 daltons in a 55°-61° GL aqueous-alcoholicmedium, separating the liquid medium which contains mucopolysaccharidesin solution and precipitating out the soluble mucopolysaccharides byalcoholic precipitation, said mucopolysaccharides having an increasedratio of anti-Xa titer to USP titer as compared to the starting heparinmucopolysaccharides being defined in claim
 1. 28. The process of claim27, which comprises recovering the alcohol-precipitatedmucopolysaccharides, subjecting an aqueous solution of saidmucopolysaccharides to gel-filtration and recovering the fraction whichhas a further increased anti-Xa titer to USP titer ratio as compared tothat of the alcohol-precipitated mucopolysaccharides.
 29. The process ofclaims 27 or 28, which comprises the further step of contacting themucopolysaccharides which have increased anti-Xa titer with antithrombinIII on a support, selectively affixing thereon the mucopolysaccharideswhich have a higher Yin-Wessler activity than the mucopolysaccharideswhich are not affixed thereon and recovering the affixedmucopolysaccharides by elution, which mucopolysaccharides have furtherincreased anti-Xa titer to USP titer ratio than the startingmucopolysaccharides.